Oxidative stress-induced cell death plays a major role in ischemia/reperfusion (I/R) injury leading to end- organ damage in brain, heart, liver, and kidney. Our previous studies identified a membrane-bound Ca2*- independent phospholipase A2, iPLA2y, in mitochondria and endoplasmic reticulum (ER) of rabbit renal proximal tubular cells (RPTC). We revealed that rabbit ER-iPLA2y has similar homology to human iPLA2y, is present in various tissues vulnerable to oxidative stress-induced injury including brain, heart, and kidney, and is cytoprotective in RPTC during oxidant-induced injury. We propose that ER-iPLA2y is protective during oxidative injury by preventing and/or repairing oxidant- induced ER lipid peroxidation, thereby preventing ER membrane disruption and Ca2+ release, and protects renal cells from injury. Because oxidative stress-induced cell death plays a major role in I/R injury, we propose that inhibition of iPLA2y in the kidney will potentiate l/R-induced Acute Renal Failure (ARF). Our preliminary studies connect the cytoprotective role of iPLA2y during oxidant-induced injury in RPTC to ER lipid peroxidation and ER Ca2+ release. Specific Aim 1 will identify the role of iPLA2y in oxidant-induced ER lipid peroxidation, membrane fluidity, and Ca2+ release using isolated ER membranes. In each set of experiments a specific inhibitor of iPLA2y, R-Bromoenol Lactone (R-BEL) and its inactive enantiomer (S- BEL, negative control) will be used to evaluate the role of iPLA2y in these changes. Specific Aim 2 will elucidate the mechanisms by which ER-iPLA2y is cytoprotective during oxidant-induced injury in RPTC. We will measure changes in cytosolic free calcium ([Ca2+f]) and ER Ca2+ stores in response to oxidative stress in the presence and absence of iPLA2y inhibition in RPTC, using a Ca2+ fluorescent probe, laser scanning confocal microscopy, and pharmacological tools that modulate ER Ca2+, and link these changes to cell death. Specific Aim 3 will test our hypothesis that iPLA2y is protective in vivo using a rabbit model of l/R- induced ARF. Rabbits will be pretreated with R-BEL or diluent control prior to I/R and differences in renal function, morphological damage, and lipid peroxidation will be assessed. Completion of these aims will elucidate the mechanism(s) by which iPLA2y is cytoprotective. These studies will help strengthen our understanding of kidney damage and failure. They may help identify therapies for renal failure, stroke, and heart attack.